Project Summary Multiple sclerosis (MS) is the most common demyelinating central nervous system (CNS) disease affecting young adults, often resulting in irreversible neurological dysfunction. B lymphocytes play a complex and critical role in MS pathology and are the target of several therapeutics in clinical trials. While monoclonal antibodies targeting the B cell surface marker CD20 (i.e. Rituximab, Ocrelizumab) dramatically reduce the annualized relapse rate and delay disability progression, not all patients respond, likely due to the heterogenous nature of MS. Currently, there is no approved technique to noninvasively visualize B cells in the CNS in order to select MS patients for anti-B cell therapies and monitor treatment responses. Positron emission tomography (PET) imaging has enormous potential to fill these gaps by providing highly specific, quantitative information by tracking B cells. Notably, PET tracers targeting CD19 and/or CD20 markers could reveal dynamic information on the pathophysiology of a wide range of B cells, ultimately enabling quantification of therapeutic effects on B cell load in the CNS and peripheral organs in real time. To date, only one PET tracer for CD20+ B cells has undergone preliminary evaluation in MS, and no PET tracers have been developed for imaging CD19, expressed on a broader range of B cells (including suspected pathogenic antibody-secreting plasmablasts and circulating plasma cells). Here, we propose to develop novel immuno-PET tracers based on clinically approved CD19 and CD20 monoclonal antibody therapeutics. Such PET tracers could undergo relatively rapid clinical translation and have immediate impact on patient stratification, dosing, and real-time therapy monitoring. Our hypothesis is that CD19 and CD20-targeted PET tracers will enable non-invasive, sensitive, and specific detection of various B cell subsets in the CNS and periphery, in the context of MS. We have previously demonstrated the feasibility of B cell specific PET imaging with [64Cu]Rituximab, which enabled in vivo detection and quantification of B cells in CNS and peripheral tissues in a murine model of MS, known as experimental autoimmune encephalomyelitis (EAE). In this proposal, we will build on our published data in addition to developing the first reported human CD19-PET tracer. We will achieve our goals through the following specific aims: 1) Develop immuno-PET tracers for imaging different B cell-subsets, 2) Assess biological effects of CD19 and CD20 imaging agents in cells and mice, and 3) Evaluate the specificity and sensitivity of CD19/CD20 PET tracers for detecting B cells in the CNS and periphery of MS mouse models in addition to their ability to predict treatment response. Completing these experiments will provide invaluable information regarding which B cell PET tracers appear most promising for clinical translation while also shedding light on the in vivo pathophysiology of B cells in MS in the brain and beyond. This proposal addresses a significant unmet clinical need and our unique approach has high potential to impact the way we study, monitor, and treat MS.